Study Of Molecular Mechanisms For The Effect Of Swine TDP2 Gene On Foot-and-Mouth Disease Virus Replication

Foot-and-Mouth Disease?FMD?is a clinically acute and high-contagious disease of cloven-hoofed animals including ruminants and pigs.FMD is caused by Foot-and-Mouth Disease Virus?FMDV?,which is a positive RNA virus from the Aphthovirus genus,Picornaviridae.VPg?Virus Protein-genome linked?is a small peptide encoded by the FMDV genome and attached to 5' end of FMDV RNA through a phosphodiesterase bond.VPg serves as primer during the FMDV genome RNA replication,thus it plays a critical role to the FMDV life circle.Tyrosyl-DNA-Phosphodiesterase-2?TDP2?is a multifunctional protein and can function as VPg unlinkase,hydrolysing the 5' tyrosyl-RNA phosphodiester bond between VPg and virus RNA.However,the relationships between TDP2 and FMDV is still unknown.In this study,the effect of TDP2 to the FMDV replication will be evaluated and the molecular mechanism for these interaction between pig TDP2 and FMDV will be studied in PK15 cellsI created TDP2 deficient PK-15 cell lines?PK-15TDP2-/-?using the CRISPR/Cas9 method,and confirmed the deletion of TDP2 genes in PK-15TDP2-/-cells through genome DNA PCR,sequencing and Western blot analyses.I then counted the growth curve of O/BY/CHA/2010 strain of FMDV serotype O and the A/GDMM/CHA/2013 strain of serotype A in PK-15TDP2-1-and wildtype PK-15 cells through the virus titer analysis method.This effort demonstrated that compared to wildtype PK-15 cells,PK-15TDP2-/-cells have a significantly lower viral titer of the O/BY/CHA/2010 and the A/GDMM/CHA/2013.At 8 hours post infection,virus titers in PK-15TDP2-/-cell lines were-200 fold lower than that in wildtype PK-15 cells.Expectedly,the O/BY/CHA/2010 FMDV strain paritialy rescued their replication ability in PK-15TDP2-/-cells when these cells are restored to express the pig TDP2 protein,implying that the TDP2 might play an important role in the FMDV replication.I further compared the entrance,viral RNA replication and translation,as well as viral release of the O/BY/CHA/2010 FMDV strain in the above PK-15 cells.This analysis suggested that the viral genomic replication and translation were inhibited,thus these viruses can't replicate rapidly in PK-15TDP2-/-cells.Such reducting of viral replication in PK-15TDP2-/-cells were partly attributed they failed to hydrolyse the 5' tyrosyl-RNA phosphodiester bond between VPg and viral RNA without using the pig TDP2 protein.I also generated two PK-15 cell lines,which one overexpressed pig TDP2 protein?PK-15TDP2-OE?and the other overexpressed pig TDP2 protein locating in Endoplasmic Reticulum?PK-15TDP2-ER?.I counted viral tilters of FMDV O/BY/CHA/2010 strain and A/GDMM/CHA/2013 strain in these cells.Interestingly,PK-15 cells and PK-15TDP2-OF cells had comparative level of the both strains of FMDV tilters during the infection course.However,PK₁5TDP2-ER cells showed significantly lower level of replication.Detailed analysis indicated that the growth curves of O/BY/CHA/2010 strain were increased from 4 h to 16 h,had a peak at 16 h and showed FMDV negative at 40 h in PK-15TDP2-ER cells.I then performed immunofluorescent staining using anti-TDP2 and anti-virus antibodies.This analysis demonstrated that the TDP2-ER protein were co-located with FMDV virus in PK-15TDP2 ER cells.These observations suggested that the inhibition of the FMDV at late stage of infection in PK-15TDP2-ER cells was possibly attributed to the hydrolyzation of the VPg protein from the FMDV genome.Moreover,I performed transcriptomic sequencing of PK-15,PK-15TDP2-OE,PK-15TDp2-ER,PK-15TDP2-/-cells,infected with or without the FMDV O/BY/CHA/2010 strain,using the Solexa technology?Hiseq-2500,Illumina?.I also identified genes showed significantly expression among these cells using the thresholds of fold change>=2 and False discovery rate<=0.001.This effort indicated that,after infected with the FMDV O/BY/CHA/2010 strain at 4 h,there were 223,709,2375 genes showing significantly expression?DEGs?in PK-15TDP2-OE,PK-15TDP2-ER,PK-15TDP2-/-cells when compared to PK-15 cells.Detailed analysis indicated that some genes involving in the immune response to the FMDV,such as PIK3,AKT3 and IL-6,showed significant expression.Pathway analysis of DEGs were enriched into steroid biosynthesis,metabolic pathway and NF-?B signal pathway.In addition,I identified 20 and 13 interactors of the pig TDP2 in PK-15 cells before or after FMDV infection through the protein pull down.Functional annotation indicated that these interactors involve into metal ion binding,RNA binding,histone and cytoskeleton formation,implying that TDP2 may affect FMDV life cycle in multiple ways.In summary,I found that the pig TDP2 might play multiple roles in the regulation of FMDV replication in host cells and built a molecular modules for the interaction between the pig TDP2 and FMDV.Moreover,I generated transcriptomic profiles of the PK-15,PK-15TDP2-PE,PK-15TDP2-ER,PK-15TDP2-/-cellc infected with or without the FMDV strain O/BY/CHA/2010.In addition,I identified 34 proteins which might involve into the regulation of FMDV replication through the TDP2 protein.These obervations would provide insights into the increasing of anti-FMDV in pig with the gene-editing technologies.